Transplantations or allografting involves the donation of material from one individual or multiple individuals, the donor(s), to another individual, the recipient. During allografting, donor cells may react to recipient cells and tissue (graft-versus-host disease), which is an unwanted reaction due to a mismatch in HLA antigens. There are several methods to identify and eliminate donor cells, typically T-lymphocytes, which react to and are activated by recipient cells. The mixed lymphocyte reaction is an ex vivo method which can be used to study the reactivity between host and donor cells. In combination with photodynamic therapy it can be used to selectively eliminate donor cells which are activated by host cells.
Such a combined identification and elimination of alloreactive T-cells is used for the manufacture of T-cell enriched donor lymphocyte preparations selectively depleted of allo-reactive T-cells towards the host. This is e.g. described in WO 01/24824 where these preparations are manufactured by mixing donor cells with γ-irradiated recipient cells in the presence of IL-2 to induce a one-way MLR of the donor cells in the direction of the recipient's mismatched major HLA-antigens. After culture period, the cells are harvested and exposed to a fluorescent photosensitive rhodamine compound, TH9402, which accumulates in all cells. After a subsequent period of incubating the cells in culture media without the presence of TH9402, the dye is preferably retained in those cells that were activated in the one-way MLR, i.e. those donor cells that reacted to the recipient's mismatched HLA-antigens. Next, the cells are exposed to light of a visible wavelength, leading to activation of TH9402 and generation of highly cytotoxic reactive oxygen radicals, thereby eliminating those donor cells that reacted to the recipient's mismatched HLA-antigens but sparing the other donor cells. Mielke et al. (2008) Blood 111: 4332-4402 describes an MLR in which expanded T-cells, generated using anti-CD3 and II-2, are co-cultured with responder cells from HLA-matched or -mismatched donors. In this study, incubations with 7.5 micromolar TH9402 give significantly better results than incubations with 5 micromolar. Perrucio et al. (2007) Blood Cells Molecules and Diseases 40: 75-83 describes an MLR process followed by photodynamic treatment with no extrusion step. Ninety to ninety-five percent of the cell population dies.
A major limitation of the method is the number of cells that can be handled within a single treatment. The concentration of cells to be treated cannot exceed one million cells/ml in the extrusion phase without compromising the quality of the final product. Therefore, in order to treat more cells one would need to increase the volume to be handled in the process. Yet, working with large volumes, e.g. one liter and larger, is difficult and impacts the robustness and reproducibility of the treatment. Photodynamic treatment of sufficient cells for one patient would require about ten illumination devices or several rounds of treatment.